Teresa Wysocka

In vitro effect of quercetin on human gastric carcinoma: targeting cancer cells death and MDR.

The benefits of plant polyphenols as chemotherapeutic agents are of great interest due to their possible anti-cancerogenic activities. Results available up to now suggest that flavonoid quercetin induces lethal effect in many types of tumours and may sensitize resistant cells to drugs. The aim of our study was to examine the effect of quercetin on human gastric carcinoma cells and to determine mode of its action. Parental EPG85-257P cell line and its daunorubicin-resistant variant EPG85-257RDB were used as cell models. Our data revealed that quercetin exerted antiproliferative impact on studied cells (with IC(50) value of 12 μM after 72 h), mainly through induction of apoptosis. In sensitive cells cytostatic drug and flavonoid had synergistic effects, in EPG85-257RDB cells quercetin acted as a chemosensitizer. Its impact on resistance mechanism involved decrease of P-glycoprotein expression, inhibition of drug transport and downregulation of ABCB1 gene expression. The results demonstrate that quercetin may be considered as a prospective drug to overcome classical resistance in gastric cancer cells.

Esters of betulin and betulinic acid with amino acids have improved water solubility and are selectively cytotoxic toward cancer cells

Betulin and betulinic acid are naturally occurring pentacyclic triterpenes showing cytotoxicity towards a number of cancer cell lines. Unfortunately they are practically insoluble in aqueous media and therefore their overall absorption index is not satisfactory. We have modified structures of both compounds by simple transformation to mono- and disubstituted esters of l-amino acids. This allowed us to achieve better water solubility without loss of the observed earlier anticancer properties. Comet assay on various cancer cell lines demonstrate that these compounds act via an apoptotic mechanism.

ABCC2 (MRP2, cMOAT) Localized in the Nuclear Envelope of Breast Carcinoma Cells Correlates with Poor Clinical Outcome

Nuclear expression of ABCC2 can be specific for lower differentiated cells and stem cells. The study aimed at examination of ABCC2 expression in breast cancers. The immunohistochemical analyses were performed on 70 samples of breast cancer. We have also studied prognostic value of the ABCC2 mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 1809 breast cancer patients. Immunohistochemical studies demonstrated that ABCC2 expression may be manifested in nuclear envelope of neoplastic cells (ABCC2n) as well as in their cell membrane and cytoplasm (ABCC2c). The univariate and multivariate analyses showed that higher expression of ABCC2n and ABCC2c was typical for cases of a shorter overall survival time. Higher ABBC2n expression was also typical for cases of a shorter disease-free survival and a shorter progression-free time. The KM plotter analysis of the prognostic value of ABCC2 mRNA expression showed that elevated ABCC2 expression was specific for cases of a shorter relapse-free survival only in the estrogen receptor-negative subgroup. The study demonstrated hat breast cancers manifest ABCC2 expression and that it is linked to a less favourable prognosis. Our results suggested that immunohistochemical tests represent a reliable way to detect prognostic value of ABCC2 expression, allowing to demonstrate differences related to subcellular localization of the protein. Cases with nuclear expression of ABCC2 manifested a more aggressive clinical course, which might reflect a less advanced differentiation of neplastic cells, resistance to the applied cytostatic drugs and tamoxifen.

Antiproliferative and pro-apoptotic effects of quercetin on human pancreatic carcinoma cell lines EPP85-181P and EPP85-181RDB.

Polyphenols are present in several edible plants and for many years induce high interest mainly due to their antioxidative and anti-inflammatory influence. At present, numerous studies are conducted on antineoplastic effects of the compounds. One of most effective biopolyphenols involves the flavonol quercetin. Our studies aimed at evaluation of antiproliferative and pro-apoptotic effects of quercetin alone and in combinations with daunorubicin on cells of human pancreatic carcinoma lines. The experiments were conducted on two cell lines, sensitive to daunorubicin EPP85-181P line, and its resistant variant EPP85-181RDB. Effect of studied substances on cell proliferation was detected using sulphorhodamine B (SRB) protein staining method. Apoptotic damage was estimated using comet and TUNEL techniques. Our data demonstrated that quercetin exerted cytotoxic action on cells of the both neoplastic cell lines in concentration-dependent manner. In the case of EPP85-181RDB cell line, quercetin seemed to sensitize resistant cells to daunorubicin. In parallel, the effect of both substances on the sensitive cell line was synergistic. Results of the studies confirmed that quercetin may probably break resistance of neoplastic cells to chemotherapy. On the other side, studied flavonol augmented action of cytostatic drug in case of sensitive tumour cells what suggest, that it might allow to decrease dosage of cytostatic drugs and reduce negative side effects of the treatment.

The new esters derivatives of betulin and betulinic acid in epidermoid squamous carcinoma treatment - In vitro studies.

BACKGROUND Betulinic acid and betulin are triterpenes with documented cytotoxic properties toward various cell lines. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications. OBJECTIVE To investigate the specific anticancer role of the newly synthesized betulin derivatives in human epidermoid carcinoma cells. METHODS In the present study we synthesized five amino acid esters of betulin. For the synthesis we selected alanine (Boc-l-Ala-OH, negative control) and four basic amino acids - natural lysine (Boc-l-Lys(Boc)-OH) and three its unnatural derivatives (Boc-l-Dap(Boc)-OH, Boc-l-Dab(Boc)-OH, and Boc-l-Orn(Boc)-OH). Boc-protected amino acids were most convenient for the synthesis. All new esters have one (betulin-l-Ala-NH2) or two free amino groups which significantly increase their solubility in water and facilitate their transport through the cell membrane. It is worth noting that the biological activity of new esters of betulin is positive correlated with the length of the side chain of l-amino acid. The highest biological activity displayed compound containing lysine side chain (Lys, -CH2-CH2-CH2-CH2-NH2). Considering the biological activity, other derivatives can be set in the following series: Orn (-CH2-CH2-CH2-NH2)>Dab (-CH2-CH2-NH2)>Dap (-CH2-NH2)>Ala (CH3)>betulin. New betulin esters were tested in normal human keratinocytes (HaCaT) and human epidermoid carcinoma cells (A431). To assess cytotoxicity, MTT test was performed after 24, 48 and 72h of incubation with the test compounds at a concentration range of 0.75-100μM. In case of apoptotic activity, a TUNEL method and comet assay were performed. Additionally expression of caspase-3 and PARP1 was evaluated immunocytochemically. RESULTS The highest cytotoxicity in cells induced skin cancer new compounds, particularly compound containing a lysine side chain (IC50=7μM) and ornithine (IC50=10μM). The highest number of apoptotic cells was observed in case incubation with compound containing Orn, Dab and Dap side chain. CONCLUSIONS The new betulin ester derivatives display enhanced antitumor activity compared to their non-modified precursors. It is worth emphasizing their specific toxicity against epidermoid carcinoma cells.

Steroid receptor status, proliferation and metallothionein expression in primary invasive ductal breast cancers.

The most important immunocytochemical prognostic and predictive factors in cases of breast cancer include estrogen receptor alpha (ER) and progesterone receptor (PgR). The present study aimed at examining the relationship between the manifestation intensity of proliferation markers (Ki-67 and nucleolar organizer regions —AgNORs) on one hand, and expression of ER and PgR on the other in a uniform group of invasive ductal breast cancers of G2 grade. Moreover, the study aimed at examining the relationship between the above mentioned markers and expression of metallothionein (MT). The studies were performed on samples of invasive ductal breast cancers of G2 grade, originating from 60 females. In paraffin sections originating from the studied cases immunocytochemical reactions were performed using monoclonal antibodies to ER, PgR, Ki-67 and MT, and silver staining was conducted to localize AgNORs. The obtained results were subjected to statistical analysis using Statistica software. Results indicate that manifestation of AgNORs does not correlate with any of the studied antigens (ER, PgR, Ki-67, MT) (p>0.05). Moreover, no relationship could be demonstrated between the intensity of MT expression and proliferation markers or steroid receptor status (p>0.05). A negative correlation was shown between the expression of ER and Ki-67 (p=0.0009). The most intense proliferative activity was demonstrated in cases of breast cancer showing PgR expression but no ER expression (p=0.015), while the lowest proliferative activity was detected in breast cancers with expression of both ER and PgR (p<0.05).

CD46 Expression is an unfavorable prognostic factor in breast cancer cases.

The membrane cofactor protein, CD46 represents a complement inhibitor, which protects autologous cells from complement-mediated cytotoxicity. On tumor cells, CD46 may exhibit the potential to protect them from immune responses of the host. The present study aimed at evaluation of prognostic significance of CD46 expression in breast cancers. The analyses were performed on 70 samples of breast cancer. Immunohistochemical reactions were performed on paraffin sections of studied tumors using monoclonal antibodies directed against CD46. Results of the immunohistochemical reactions and of clinical observations were subjected to statistical analysis. Multivariate analysis showed that expression of CD46 and involvement of lymph nodes represent independent risk factors for disease-free survival and overall survival. Kaplan-Meier analysis showed that patients with tumors negative for CD46 have an increased progression-free time and overall survival time as compared with patients with the CD46-positive tumors. The study demonstrates that breast cancers manifest CD46 expression and that it is linked to a less favorable prognosis.

Electroporation-induced changes in normal immature rat myoblasts (H9C2)

Application of a high electric field causes an electric shock to the heart. This is utilized in defibrillation to reestablish normal contraction rhythms during dangerous arrhythmias or in cardiac arrest. If shock-induced transmembrane potentials are large enough, they can cause tissue destruction due to irreversible electroporation (EP). Also electrochemotherapy of nearby tissues may have an adverse effect on the heart. Herein, we present experimental data on effects of electroporation in culture of cardiac cells (H9C2). The electric field was applied in short pulses of 25-3250 V/cm, 50 µs each. The viability of cells was tested by MTT assay after 24 hours. For detection of DNA fragmentation, associated with apoptosis, alkaline and neutral comet assays were performed after EP. Additionally phase contrast images of cells obtained directly after EP were analyzed. Although cell images indicated disruption of cell membranes after EP with high intensities, only a few percent of apoptotic cells and no necrotic effects in the cell nucleus could be observed in comet assay tests performed 2 hours post EP. MTT viability test showed that pulse intensities above 375 V/cm are destructive for myocytes viability.

Oxidative modulation of marcaine and lekoptin in H9C2 rat myoblasts

AbstractAim:The cytotoxicity of marcaine was estimated in combination with a calcium channel blocker. In addition, the influence of marcaine and marcaine plus lekoptin on a model system using the H9C2 cardiac cell line was investigated.Methods:Cells were incubated for five hours with marcaine, lekoptin, or with both drugs simultaneously. Apoptotic cells were detected using the TUNEL assay and the alkaline comet assay. Mitochondrial cell function after drug uptake was examined using the MTT assay. The concentration of MDA (malondialdehyde) — the final product of fatty-acid peroxidation, was quantified spectrophotometrically. The expression of glutathione S-transferase π (GST-π) was detected by immunofluorescence (IF) and Western blotting (WB) and inducible nitric oxide synthase (iNOS) was assessed by immunocytochemical staining (ABC).Results:Incubation with marcaine resulted in the highest number of apoptotic cells. After incubation with both marcaine and lekoptin, moderate damage to cells (54.2%±1.775% of DNA destruction) was observed. The highest levels of iNOS and GST-π expression were observed in cells treated with marcaine and marcaine plus lekoptin. The characteristic nuclear GST-π expression was observed in cells treated with both drugs.Conclusion:Lekoptin stimulated cells to proliferate. Marcaine caused membrane damage and ultimately cell death.

Effects of electrophotodynamic therapy in vitro on human melanoma cells--melanotic (MeWo) and amelanotic (C32).

Photodynamic therapy has been considered ineffective for melanomas because of the competition between the absorbance of melanin from the melanoma and the absorbance of photosensitizers at the photosensitizer excitation light wavelength. Melanomas show considerable heterogeneity and resistance to phototherapy. The effectiveness of photodynamic therapy could be intensified by electroporation for enhanced transport of a photosensitizer by transient pores in the membrane. In this study, photodynamic therapy combined with electroporation was tested in vitro on the human melanoma cell lines melanotic melanoma (MeWo) and amelanotic melanoma (C32). Control experiments were conducted on human keratinocytes (HaCaT). Photofrin was used as a photosensitizer. Photosensitizer distribution, cloning efficacy test, comet assay, and assessment of apoptotic proteins were performed. Melanin levels were determined before and after photodynamic therapy. The experiments indicated that electroporation effectively supports the photodynamic method. It was found that photodynamic therapy with electroporation efficiently induces apoptosis in melanotic and amelanotic melanoma cells.